Ribonucleic acid (hereinafter RNA) is a complex polymer occurring in living organisms. Certain enzymes are capable of splitting the RNA molecule into smaller subunits known as nucleotides. The 5'-nucleotides are of particular interest because of their ability to improve the richness and intensity of flavors without themselves contributing to taste of a food product. For this reason, there is considerable commercial interest in the preparation of the enzyme which hydrolyzes RNA to form 5'-nucleotides. This enzyme is known as 5'-phosphodiesterase.
It is well known that rapidly proliferating parts of germinating seeds, such as the rootlets and stems, contain 5'-phosphodiesterase. However, these materials contain other enzymes which are extracted along with the 5'-phosphodiesterase. Some of these other enzymes in the crude extract cause further hydrolysis of 5'-nucleotides. This makes the crude extracts unsuitable for use in preparing 5'-nucleotides from RNA.
In U.S. Pat. No. 3,304,238, there is disclosed a method for preparing an aqueous enzyme medium capable of forming 5'-ribonucleotides from RNA. According to this disclosure, rapidly proliferating, substantially noncomminuted seed parts are heated in water to from about 70.degree. to 85.degree. C. for several minutes. Malted barley rootlets are included in the list of seed parts used. The crude mixture of solid and liquid is used to hydrolyze RNA.
In U.S. Pat. No. 3,459,637, ground rootlets or sprouts are extracted with water and the filtered extracts are used to hydrolyze RNA. In order to obtain 5'-nucleotides from RNA using this crude extract, it is necessary to carry out the reaction with RNA at a high pH, between 8.5 and 9.5. Zinc ions are added to accelerate the reaction.
U.S. Pat. No. 3,516,907 discloses a method for obtaining 5'-nucleotides by reacting nucleic acids with a 5'-phosphodiesterase active extract. This extract is obtained by extracting plant material, such as barley sprouts, with water. The crude extract could be further purified by gel fractionation or selective precipitation with a solvent. Heat treatment of the vegetable extract, preferably in the presence of a heavy metal salt, is also disclosed.
A method for producing a yeast extract of improved flavor is disclosed in U.S. Pat. No. 4,303,680. The method involves treating yeast RNA with a crude 5'-phosphodiesterase enzyme solution. The enzyme solution is obtained by extracting broken malt roots with water and then heating the clear filtrate at a temperature of 60.degree. to 65.degree. C. for 5 to 10 minutes.
Although the foregoing references disclose general methods for extracting an enzyme solution having phosphodiesterase activity from plant parts, the general methods are only suitable for use on a laboratory scale. Furthermore, these crude extracts tend to lose activity on storage unless they are kept in a frozen state.
We have now discovered a process for producing a 5'-phosphodiesterase enzyme preparation with low 5'-nucleotidase content which is adaptable to large-scale commercial production. This process is capable of producing both liquid and solid enzyme preparations which have excellent storage stability.
The present process is based on two discoveries. The tightly bound to ground barley malt sprouts than is the desired 5'-phosphodiesterase. The second discovery is that when aqueous slurries of the ground sprouts are heated, the ratio of 5'-phosphodiesterase to 5'-nucleotidase increases more rapidly if the solid material is first removed from the slurry.